The human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), are leading causes of herpesvirus-associated morbidity and mortality worldwide. An understanding of the molecular mechanisms used by gammaherpesviruses to undergo productive replication, and promote latency and reactivation is key to the rational design of clinical therapeutics. The tegument proteins that are delivered with the virion into a newly infected cell play critical roles during both early and late stages of virus production and are an integral, yet understudied component of chronic herpesvirus infection and associated disease. The role of tegument proteins are difficult to ascertain for EBV and KSHV given their strict host tropism and the lack of robust virus replication in cell culture. Here, we propose to use murine gammaherpesvirus 68 (MHV68) to disect the function of gammaherpesvirus tegument proteins during virus replication and latency. Exciting preliminary results indicate that the ORF75A and ORF75B tegument proteins have distinct roles during early and late stages of virus replication, and these functions impair the ability of the ORF75B null virus to establish latency in the spleens of infected mice. The central hypothesis of this proposal is that the ORF75A and ORF75B tegument proteins of MHV68 have separable roles during virus replication upon de novo productive infection that will impair latency and reactivation in vivo. This proposal describes innovative, high-impact experimental approaches to elucidate the contribution of ORF75 tegument proteins to gammaherpesvirus pathogenesis. In Aim 1, we will discern the role of ORF75A and ORF75B by identifying the defects in virus replication upon conditional ablation of the proteins in the tegument of incoming particles or ablation of newly synthesized protein. In Aim 2, we will use the intraperitoneal route of infection to circumvent lung replication defects in the acute phase upon the intranasal route of inoculation to directly examine the role of ORF75A and ORF75B in the establishment of latency and reactivation from latency in the B cells and macrophages of infected mice. We expect to uncover novel functions that will establish a framework for further mechanistic investigations of ORF75 and provide new insight into the role of tegument proteins as determinants of chronic gammaherpesvirus infection and KSHV- and EBV-associated diseases. PUBLIC HEALTH RELEVANCE: Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus have strong etiological links to numerous cancers, neoplasms, and disease in immunocompetent and immunocompromised patients, and as such, are significant pathogens worldwide. The murine gammaherpesvirus-68 model system provides the opportunity to examine multiple aspects of productive replication upon de novo infection culture and study disease development in the whole animal, enabling rapid validation of the biological relevance of virus-host interactions identified in cell culture. The information from these studies wil elucidate the key role that the viral tegument proteins ORF75A and ORF75B play in murine gammaherpesvirus pathogenesis and may lead to a translational impact on the prevention and treatment of virus-associated diseases. !